The smooth muscle cell is a cell which performs a unique combination of both muscle and nonmuscle functions as its major roles, and the actin in these cells is organized into specific structures to carry out these tasks. Although smooth muscle cells from different tissues have as a common function the generation of contractile force, the ratios of the three electrophoretically distinct actin isomers found within these cells differ from tissue to tissue. The aims of this proposal are to determine the significance of varying ratios of actin isomers in smooth muscle tissues as well as to elucidate the nature of the elements controlling the organization and assembly of actin-containing structures in smooth muscle cells. We will purify the actins from three mammalian smooth muscle tissues, aorta, uterus, and vas deferens. During these purification procedures, we will check for selective compartmentalization of the individual actin isomers within the cell. Attempts will then be made using preparative isoelectric focusing, polyacrylamide gel electrophoresis and selective immunoadsorption to resolve the individual components of the pure multi-isomer mixtures. The pure mixtures and, hopefully, the pure actin isomers will be examined on a quantitative basis for their ability to activate the myosin ATPase activity and for their efficiency of polymerizing and depolymerizing under defined circumstances. We will also isolate factors which influence actin polymerization by use of a microultracentrifugation assay. These experiments will help define differences in contractility seen between various smooth muscle tissues. We will isolate and purify the Ca plus 2 sensitive myosin light chain kinase from smooth muscle and examine the relationship between its Ca plus 2 binding and phosphorylating activities. This will also provide a more sensitive assay for assaying the actomyosin interaction. To further judge the appropriateness of using aorta smooth muscle cell culture systems for studying smooth muscle pathogenic states, we will characterize these cultures from avian and mammalian systems as a function of culture age with respect to the following parameters: 1) ratios of the three actin isomers, 2) types of myosin synthesized 3) types of Ca plus 2 control of actomysosin present. We will compare our results with those obtained from freshly prepared smooth muscle tissue to see if significance variations in (Text Truncated - Exceeds Capacity)